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US8710005B2 - Neuronal differentiation-inducing peptide and use thereof - Google Patents

Neuronal differentiation-inducing peptide and use thereof Download PDF

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US8710005B2
US8710005B2 US13/258,788 US201013258788A US8710005B2 US 8710005 B2 US8710005 B2 US 8710005B2 US 201013258788 A US201013258788 A US 201013258788A US 8710005 B2 US8710005 B2 US 8710005B2
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amino acid
peptide
seq
acid sequence
sequence
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US20120035112A1 (en
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Tetsuhiko Yoshida
Nahoko Kobayashi
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Toagosei Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a neuronal differentiation-inducing peptide and the use thereof. More particularly, the invention relates to a neuronal differentiation inducer containing such a peptide as the active ingredient.
  • One challenge in the field of regenerative medicine is the regeneration of nerve cells. It is hoped, for example, that the regeneration of nerve cells using neuronal stem cells or embryonic stem cells (ES cells) will lead to treatments for central nervous system diseases such as Parkinson disease and Alzheimer disease (Patent Document 1).
  • ES cells embryonic stem cells
  • embryonic and other stem cells are difficult to acquire (collect).
  • stem cells are implanted directly to the affected area, substantially no differentiation to nerve cells occurs and engraftment is difficult. Even in cases where such cells do take, few if any end up differentiating to glia cells.
  • Somatic stem cells such as neuronal stem cells, skin stem cells and adipose stem cells are stem cells that are relatively easy to acquire. Were it possible to differentiate such stem cells to nerve cells, their utility in the healthcare industry would be high. However, a method for inducing the differentiation of nerve cells from these somatic stem cells in a short period of time and at a high efficiency has yet to be established. Hence, there exists a desire for such a method to be established; that is, for the development of a neuronal differentiation inducer suitable for such a purpose.
  • Patent Document 2 discloses a neuronal differentiation inducer containing a pyrrolidone derivative as the active ingredient, but it does not disclose the effects of inducing the differentiation of nerve cells from somatic stem cells.
  • Patent Document 3 describes a peptide (VHL peptide) which is capable of inducing neuronal differentiation from neuronal stem cells or skin stem cells.
  • the present invention was conceived by taking an approach which differs from the approach taken in developing neuronal differentiation inducers containing conventional chemical substances such as that described in Patent Document 2.
  • the object of this invention is to provide a synthetic peptide having a higher neuronal differentiation-inducing activity than known neuronal differentiation-inducing peptides like those mentioned in Patent Document 3.
  • a further object is to provide neuronal differentiation inducers (pharmaceutical compositions) in which such peptides serve as the active ingredients.
  • a still further object is to provide a method of producing nerve cells using such peptides, and a method of inducing the development of nerve cells.
  • the neuronal differentiation-inducing peptide provided by the invention is a synthetic peptide which has been artificially designed and does not exist alone by itself as a neuronal differentiation-inducing peptide in the natural world.
  • amyloid precursor protein The inventors have closely investigated the amyloid precursor protein.
  • amyloid precursor protein APP
  • amyloid precursor protein typically composed of 40 or 42 amino acid residues
  • amyloid precursor protein typically composed of 40 or 42 amino acid residues
  • the amyloid precursor protein may be considered the starting substance for Alzheimer disease.
  • the inventors have paid particularly close attention to the signal peptide of this amyloid precursor protein.
  • Non-Patent Document 2 is an amino acid sequence which takes part in extracellular to intranuclear (typically, the nucleolus) peptide migration, and that by using a synthetic peptide constituted so as to include this amino acid sequence, the efficiency of differentiation from stem cells to nerve cells can be greatly increased.
  • NoLS nucleolar localization signal
  • the neuronal differentiation-inducing peptide disclosed herein is a peptide which is capable of inducing the differentiation of at least one type of stem cell to a nerve cell (Hereinafter, the term “neuronal differentiation-inducing peptide” means this peptide).
  • the neuronal differentiation-inducing peptide of the present invention includes an amino acid sequence constituting a signal peptide in amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • the neuronal differentiation-inducing peptide of the invention includes an N-terminal side partial amino acid sequence which is part of the amino acid sequence constituting the above signal peptide and is composed of at least six consecutive amino acid residues counting from the N-terminal amino acid residue of this sequence.
  • the neuronal differentiation-inducing peptide of the invention includes a C-terminal side partial amino acid sequence which is part of the amino acid sequence constituting the above signal peptide and is composed of at least five consecutive amino acid residues counting from the C-terminal amino acid residue of this sequence.
  • the amino acid sequence constituting a signal peptide in amyloid precursor protein (APP) and the partial amino acid sequence in such a signal peptide sequence (that is, the N-terminal side partial amino acid sequence or C-terminal side partial amino acid sequence) which is included in the neuronal differentiation-inducing peptide disclosed herein are collectively referred to as the “APP signal peptide-related sequence.”
  • the left side is always the N-terminal side and the right side is always the C-terminal side.
  • the amyloid precursor protein signal peptide is the amino acid sequence MLPGLALLLLAAWTARA (SEQ ID NO: 2) or MLPSLALLLLAAWTVRA (SEQ ID NO: 3)
  • the APP signal peptide-related sequence is the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or an N-terminal side partial amino acid sequence which is part of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 and is composed of at least six consecutive amino acid residues counting from the N-terminal amino acid residue of the sequence, or a C-terminal side partial amino acid sequence which is part of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 and is composed of at least five consecutive amino acid residues counting from the C-terminal amino acid residue of the sequence.
  • These APP signal peptide related sequences may include, aside from the respective amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3,
  • the peptide is an artificially synthesized peptide which further includes the following amino acid sequence constituting a nucleolar localization signal:
  • the peptide includes the APP signal peptide-related sequence on the N-terminal side of the amino acid sequence constituting the nucleolar localization signal.
  • the total number of amino acid residues constituting the peptide is 50 or less.
  • Preferred examples of neuronal differentiation-inducing peptides provided by the invention include synthetic peptides composed of the amino acid sequence indicated in any one of the sequence numbers selected from among SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31.
  • the invention provides a neuronal differentiation inducer which is capable of inducing the differentiation of at least one type of stem cell into a nerve cell, and which includes any one of the neuronal differentiation-inducing peptides disclosed herein and at least one pharmaceutically acceptable carrier.
  • the neuronal differentiation inducer disclosed herein includes as a neuronal differentiation-inducing peptide an artificially synthesized peptide containing an amino acid sequence constituting a signal peptide in amyloid precursor protein (APP) or containing, as an APP signal peptide-related sequence, an N-terminal side partial amino acid sequence which is part of the amino acid sequence constituting the signal peptide and is composed of at least six consecutive amino acid residues counting from the N-terminal amino acid residue of the sequence or a C-terminal side partial amino acid sequence which is part of the amino acid sequence constituting the signal peptide and is composed of at least five consecutive amino acid residues counting from the C-terminal amino acid residue of the sequence.
  • APP amyloid precursor protein
  • the signal peptide of the amyloid precursor protein has the following amino acid sequence: MLPGLALLLLAAWTARA (SEQ ID NO: 2) or MLPSLALLLLAAWTVRA (SEQ ID NO: 3), and the APP signal peptide-related sequence is the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or an N-terminal side partial amino acid sequence which is part of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 and is composed of at least six consecutive amino acid residues counting from the N-terminal amino acid residue of the sequence, or a C-terminal side partial amino acid sequence which is part of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 and is composed of at least five consecutive amino acid residues counting from the C-terminal amino acid residue of the sequence.
  • the neuronal differentiation-inducing peptide included in the neuronal differentiation inducer is preferably an artificially synthesized peptide which additionally includes the following amino acid sequence constituting a nucleolar localization signal:
  • the neuronal differentiation-inducing peptide included in the neuronal differentiation inducer prefferably includes the APP signal peptide-related sequence on the N-terminal side of the amino acid sequence constituting the nucleolar localization signal, and especially preferable for the total number of amino acid residues constituting the peptide to be 50 or less.
  • Preferred examples of the neuronal differentiation-inducing peptide included in the neuronal differentiation inducer include synthetic peptides composed of the amino acid sequence indicated in any one of the sequence numbers selected from among SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31.
  • the invention provides various methods which employ the neuronal differentiation inducer or neuronal differentiation-inducing peptide disclosed herein.
  • the invention provides a method of producing nerve cells from at least one type of cell material.
  • This method is characterized by preparing any one of the synthetic peptides disclosed herein or a neuronal differentiation inducer (pharmaceutical composition) containing the synthetic peptide, and supplying the peptide or neuronal differentiation inducer to the cell material.
  • the invention also provides a method of generating nerve cells in a living organism or in living tissue.
  • This method is characterized by preparing any of the synthetic peptides disclosed herein or a neuronal differentiation inducer (pharmaceutical composition) containing the synthetic peptide, and supplying the synthetic peptide or the neuronal differentiation inducer (pharmaceutical composition) containing the synthetic peptide to a living organism or to living tissue which has been temporarily or permanently removed from a living organism.
  • the invention additionally provides an artificially designed polynucleotide which does not exist in nature and which includes a nucleotide sequence encoding any one of the synthetic peptides disclosed herein and/or a nucleotide sequence complementary to such a sequence (e.g., polynucleotides substantially composed of these sequences).
  • polynucleotides examples include polynucleotides containing a nucleotide sequence encoding an amino acid sequence shown in any one of SEQ ID NOS: 4 to 31 and/or a nucleotide sequence complementary to such a sequence (e.g., polynucleotides substantially composed of these sequences).
  • the neuronal differentiation-inducing peptide of the invention is, as mentioned above, a synthetic peptide having a simple construction which includes an APP signal peptide-related sequence (preferably an amino acid sequence composed of an APP signal peptide-related sequence and a nucleolar localization signal (NoLS)), it can easily be produced. Hence, the desired amount of peptide (and, by extension, the neuronal differentiating inducer) can easily be prepared.
  • an APP signal peptide-related sequence preferably an amino acid sequence composed of an APP signal peptide-related sequence and a nucleolar localization signal (NoLS)
  • neuronal differentiation-inducing peptide neuronal differentiation inducer
  • non-neuronal cells typically somatic stem cells such as neuronal stem cells, adipose stem cells and skin stem cells, or embryonic stem cells
  • nerve cells nerve cells
  • nerve cell generation can be achieved.
  • FIG. 1 is a fluorescence micrograph (image) obtained by adding a sample peptide (Sample 1) to a culture solution of mouse neuronal stem cells to a concentration within the solution of 0.5 ⁇ M and culturing for 7 days, then examining the state of neuronal differentiation by the cultured cells; the image was prepared by merging a differential interference contrast (DIC) image, a DAPI nuclear stain image, and a fluorescence image showing the results of an investigation by an immune antibody method using fluorochrome-labeled anti-tubulin antibody.
  • DIC differential interference contrast
  • FIG. 2 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 2 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 3 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 3 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 4 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 4 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 5 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 5 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 6 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 6 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 7 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 7 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 8 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 8 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 9 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 9 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 10 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 10 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 11 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 11 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 12 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 12 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 13 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 13 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 14 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 14 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 15 is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by cells which, aside from using Sample 15 as the peptide, were cultured under the same conditions and materials as when the fluorescence micrograph (image) of FIG. 1 was taken.
  • FIG. 16 is a fluorescence micrograph (image) obtained by culturing mouse neuronal stem cells in a neuronal differentiation culture (to which a neuronal differentiation-inducing peptide was not added) for 7 days, then examining the state of the cultured cells; the image was prepared by merging a differential interference contrast (DIC) image, a DAPI nuclear stain image, and a fluorescence image showing the results of an investigation by an immune antibody method using fluorochrome-labeled anti-tubulin antibody.
  • DIC differential interference contrast
  • FIG. 17 is a fluorescence micrograph (image) obtained by culturing mouse neuronal stem cells in an ordinary growth culture (to which a neuronal differentiation-inducing peptide was not added) for 7 days, then examining the state of the cultured cells; the image was prepared by merging a differential interference contrast (DIC) image, a DAPI nuclear stain image, and a fluorescence image showing the results of an investigation by an immune antibody method using fluorochrome-labeled anti-tubulin antibody.
  • DIC differential interference contrast
  • artificially synthesized neuronal differentiation-inducing peptide is not a peptide chain which by itself independently exists stably in the natural world, but refers to a peptide fragment manufactured by artificial chemical synthesis or biosynthesis (i.e., genetic engineering-based production) and is capable of existing stably within a predetermined system (e.g., a composition making up a neuronal differentiation inducer).
  • peptide is a term which denotes an amino acid polymer having a plurality of peptide bonds. Although a peptide is not limited by the number of amino acid residues included on the peptide chain, the total number of amino acid residues is typically 100 or less, and preferably 50 or less.
  • amino acid residue is a term which includes the N-terminal amino acid and the C-terminal amino acid of a peptide chain.
  • an amino acid sequence that has been partially modified (modified amino acid sequence),” as used with respect to a particular amino acid sequence refers to an amino acid sequence which was formed by substituting, deleting and/or adding (inserting) one or a plurality of (e.g., two or three) amino acid residues without a loss in the neuronal differentiation-inducing ability of the particular amino acid sequence.
  • sequences that arise due to conservative amino acid replacement by one or a plurality of (typically two or three) amino acid residues e.g., a sequence in which a basic amino acid residue has been replaced with another basic amino acid residue
  • sequences that arise when one or a plurality of (typically two or three) amino acid residues have been added (inserted) to or deleted from a particular amino acid sequence are typical examples encompassed by “an amino acid sequence that has been partially modified (modified amino acid sequence)” in this specification.
  • polynucleotide is a term denoting a polymer (nucleic acid) in which a plurality of nucleotides are linked by phosphodiester bonds, and is not limited by the number of nucleotides.
  • polynucleotide encompasses DNA fragments and RNA fragments of various lengths.
  • artificially designed polynucleotide refers to a polynucleotide having a nucleotide chain (full length) which does not independently exist in the natural world but has been artificially synthesized, either by chemical synthesis or biosynthesis (i.e., genetic engineering-based production).
  • the inventors have discovered that relatively short peptides synthesized so as to include an amino acid sequence corresponding to the signal peptide of the amyloid precursor protein (APP) produced in the nerve cells of the brains of mammals such as humans, chimpanzees, crab-eating macaques, mice and rats are able to exhibit a remarkable neuronal differentiation-inducing activity.
  • APP amyloid precursor protein
  • Non-Patent Document 1 is cited above as a review article
  • the neuronal differentiation of at least one type of stem cell e.g., various somatic stem cells, embryonic stem cells, synthetic pluripotent stem cells
  • stem cell e.g., various somatic stem cells, embryonic stem cells, synthetic pluripotent stem cells
  • amino acid sequences of the amyloid precursor protein signal peptides preferably used in practicing the invention are shown respective in SEQ ID NO: 2 and SEQ ID NO: 3.
  • MLPGLALLLLAAWTARA is a signal peptide sequence composed of 17 amino acid residues in the amyloid precursor protein produced in the nerve cells of the brains of humans, chimpanzees and crab-eating macaques.
  • MLPSLALLLLAAWTVRA is a signal peptide sequence composed of 17 amino acid residues in the amyloid precursor protein produced in the nerve cells of the brains of mice and rats.
  • amino acid sequence (composed of 17 amino acid residues) indicated in above SEQ ID NO: 2 or SEQ ID NO: 3 may be employed directly as the APP signal peptide-related sequence.
  • the N-terminal side partial amino acid sequence composed of at least six consecutive amino acid residues counting from the N-terminal amino acid residue on the signal peptide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, that is, the N-terminal side partial amino acid sequence wherein the position 1 methionine residue to the position 6 alanine residue counting from the N-terminal amino acid residue are essential and amino acid residues to the C-terminal side therefrom are optional, can be used as the APP signal peptide-related sequence.
  • Specific examples of the N-terminal side partial amino acid sequence include the following.
  • a C-terminal side partial amino acid sequence composed of at least five consecutive amino acid residues counting from the C-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 2 or SEQ ID NO: 3, that is, the C-terminal side partial amino acid sequence wherein the position 13 tryptophan residue to the position 17 (C-terminal) alanine residue counting from the N-terminal amino acid residue are essential and amino acid residues to the N-terminal side therefrom are optional, can be used as the APP signal peptide-related sequence.
  • Specific examples of the C-terminal side partial amino acid sequence include the following.
  • the designed neuronal differentiation-inducing peptide may be a peptide composed solely of the above APP signal peptide-related sequence or a modified amino acid sequence thereof
  • an amino acid sequence which constitutes a protein transduction domain is preferred.
  • Preferred examples are shown in SEQ ID NOS: 33, 34 and 35.
  • SEQ ID NO: 33 shows the amino acid sequence of the protein transduction domain included in the TAT of HIV, and a peptide composed of this sequence.
  • SEQ ID NO: 34 shows the amino acid sequence of a protein transduction domain (PTD4) obtained by modifying the above TAT, and a peptide composed of this sequence.
  • SEQ ID NO: 35 shows the ANT-related amino acid sequence of the fruit fly (Drosophila) variant Antennapedia.
  • KKRTLRKNDRKKR is especially preferred.
  • Non-Patent Document 2 a peptide containing an amino acid sequence composed of the amino acid sequence shown in above SEQ ID NO: 1, known as a nucleolar localization signal (NoLS), and other target amino acid sequences (relatively short sequences which can be related to some other function; i.e., peptide motifs) is synthesized and added to eukaryotic cells being cultured, the peptide is able to pass through the cell membrane of the target cells at a high efficiency, and also is able to pass through the nuclear membrane at a high efficiency.
  • NoLS nucleolar localization signal
  • the present invention by constructing (synthesizing) an artificial peptide obtained by combining a target APP signal peptide-related sequence (peptide motif related to neuronal differentiation induction) and the amino acid sequence shown in SEQ ID NO: 1 above (also referred to below as “nucleolar localization signal-related sequence”) and adding the artificial peptide to a target eukaryotic cell, enables the artificial peptide to be efficiently transferred from the eukaryotic cell exterior (outside the cell membrane) into the nucleus (preferably the nucleolus).
  • a target APP signal peptide-related sequence peptide motif related to neuronal differentiation induction
  • amino acid sequence shown in SEQ ID NO: 1 above also referred to below as “nucleolar localization signal-related sequence”
  • At least one amino acid residue of the neuronal differentiation-inducing peptide provided by this invention is amidated.
  • the structural stability e.g., the protease resistance
  • the total number of amino acid residues making up the peptide chain is 100 or less, and preferably 50 or less.
  • the chemical synthesis of such peptides having a short chain length is easy, enabling neuronal differentiation-inducing peptides to be readily provided.
  • the conformation of the peptide is not subject to any particular limitation, so long as the peptide exhibits a neuronal differentiation-inducing ability in the environment in which it is used. However, a straight-chain or helical configuration is preferred because the peptide does not readily become an immunogen (antigen). A peptide having such a shape does not readily form an epitope.
  • neuronal differentiation-inducing peptides suitable for a neuronal differentiation inducer.
  • the APP signal peptide-related sequence it is desirable for the APP signal peptide-related sequence to account for a proportion of the entire amino acid sequence (i.e., the number of amino acid residues constituting the APP signal peptide-related sequence portion, as a percentage of the total number of amino acid residues constituting the peptide chain) which, although not subject to any particular limitation so long as the neuronal differentiation-inducing activity is not lost, is at least 20%, and preferably from 30 to 50%.
  • the neuronal differentiation-inducing peptide of the invention is preferably such that all the amino acid residues are L-type amino acids. However, to the extent that the neuronal differentiation-inducing activity is not lost, some or all of the amino acid residues may be substituted with D-type amino acids.
  • the neuronal differentiation-inducing peptide of the invention may include in portions thereof sequences which cannot be included in the APP signal peptide-related sequences and the nucleolar localization signal-related sequences.
  • sequences which are able to retain the three-dimensional shape (typically, the straight-chain shape) of the APP signal peptide-related sequence portion in the peptide chain are preferred as such partial sequences.
  • An illustrative example is a linker sequence (hinge region) which links together an APP signal peptide-related sequence portion and a nucleolar localization signal-related sequence portion.
  • linker sequences include those composed of about one to nine (e.g., one, two or three) glycine residues and/or serine residues (see the subsequently described working examples).
  • neuronal differentiation-inducing peptides disclosed herein those having a relatively short peptide chain can easily be produced according to a common chemical synthesis process.
  • a known solid-phase synthesis process or liquid-phase synthesis process use may be made of a known solid-phase synthesis process or liquid-phase synthesis process.
  • a solid-phase synthesis process which employs t-butyloxycarbonyl (Boc) or 9-fluorenylmethoxycarbonyl (Fmoc) as the amino group-protecting group is preferred.
  • the neuronal differentiation-inducing peptide having a number of amino acid residues of 100 or less (especially 50 or less) disclosed herein can easily be synthesized as a peptide chain having the desired amino acid sequence and modifying (e.g., C-terminal amidating) portions by a solid-phase synthesis process using a commercial peptide synthesizer (available from, for example, Intavis AG or Applied Biosystems).
  • the neuronal differentiation-inducing peptide may be biosynthesized by a genetic engineering technique. This approach is preferred in cases where a polypeptide having a relatively long peptide chain is produced. That is, the DNA of a nucleotide sequence (including the ATG initiation codon) which codes for the amino acid sequence of the desired neuronal differentiating-inducing peptide is synthesized. Then, a recombinant vector having an expression gene construct composed of this DNA and various regulatory elements (including promoters, ribosome binding sites, terminators, enhancers, and various cis-elements which control the expression level) for expressing this amino acid sequence within a host cell is constructed in accordance with the host cell.
  • various regulatory elements including promoters, ribosome binding sites, terminators, enhancers, and various cis-elements which control the expression level
  • this recombinant vector is inserted into given host cells (e.g., yeasts, insect cells, plant cells, mammalian cells), and the host cells or tissue or individuals containing those cells are cultured under specific conditions.
  • host cells e.g., yeasts, insect cells, plant cells, mammalian cells
  • the target polypeptide can be expressed and produced intracellularly.
  • the target neuronal differentiation-inducing peptide can be obtained.
  • a fused protein expression system may be employed for efficient large-volume production within host cells. That is, a gene (DNA) coding for the amino acid sequence of the target neuronal differentiation-inducing peptide is chemically synthesized, and the synthesized gene is introduced to a preferred site on a suitable fused protein expression vector (a glutathione S-transferase (GST) fused protein expression vector such as the pET series available from Novagen and the pGEX series available from Amersham Bioscience).
  • GST glutathione S-transferase
  • the host cells typically, Escherichia coli
  • the resulting transformant is cultured, thereby producing the target fused protein. This protein is then extracted and purified.
  • the purified fused protein thus obtained is cleaved with a specific enzyme (protease), and the liberated target peptide fragments (the designed neuronal differentiation-inducing peptide) are recovered by a method such as affinity chromatography.
  • the neuronal differentiation-inducing peptide of the invention may be produced by using such a conventional, known fused protein expression system (e.g., the GST/His system available from Amersham Bioscience may be used).
  • the target polypeptide may be synthesized in vitro by constructing template DNA for an acellular protein synthesis system (i.e., a synthesized gene fragment having a nucleotide sequence which codes for the amino acid sequence of the neuronal differentiation-inducing peptide) and, using the various compounds required for peptide synthesis (e.g., ATP, RNA polymerase, amino acids), employing an acellular protein synthesis system.
  • an acellular protein synthesis system i.e., a synthesized gene fragment having a nucleotide sequence which codes for the amino acid sequence of the neuronal differentiation-inducing peptide
  • the various compounds required for peptide synthesis e.g., ATP, RNA polymerase, amino acids
  • PROTEIOSTM a wheat germ cell-free protein synthesis kit available from Toyobo Co., Ltd. (Japan), is commercially available.
  • the target neuronal differentiation-inducing peptide can easily be synthesized and produced by an acellular protein synthesis system in accordance with the amino acid sequence.
  • the neuronal differentiation-inducing peptide of the invention can be easily produced based on the Puresystem® from Post Genome Institute Co., Ltd.
  • a single-strand or double-strand polynucleotide containing a nucleotide sequence coding for the neuronal differentiation-inducing peptide disclosed herein and/or a nucleotide sequence complementary thereto can easily be produced (synthesized) by a hitherto known method. That is, by selecting codons corresponding to the respective amino acid residues making up the designed amino acid sequence, the nucleotide sequence corresponding to the amino acid sequence of the neuronal differentiation-inducing peptide is easily determined and provided.
  • the target double-strand DNA can be obtained using various enzymatic means of synthesis (typically, PCR).
  • the polynucleotide provided by the invention may be in the form of DNA or in the form of RNA (e.g., mRNA).
  • the DNA may be provided as double-stranded DNA or as single-stranded DNA. When provided as a single strand, it may be either a coding strand (sense strand) or an anticoding strand (antisense strand) which is the sequence complementary thereto.
  • the polynucleotide provided by the invention may, as described above, be used as a material for constructing a recombinant gene (expression cassette) for producing the neuronal differentiation-inducing peptide, either in various host cells or in an acellular protein synthesis system.
  • a polynucleotide having a nucleotide sequence which codes for a neuronal differentiation-inducing peptide having a novel amino acid sequence and/or a nucleotide sequence complementary to such a sequence For example, artificially designed polynucleotides which include (or are substantially composed of) nucleotide sequences coding for the respective amino acid sequences of SEQ ID NOS: 1 to 35 in which the total number of amino acid residues making up the peptide chain is 50 or less (preferably 40 or less), and/or nucleotide sequences complementary thereto, are provided.
  • Preferred neuronal differentiation-inducing peptides of the invention have a high neuronal differentiation-inducing activity on at least one type of cell. For this reason, they can be advantageously used as the active ingredient in a neuronal differentiation inducer.
  • the neuronal differentiation-inducing peptide included in the neuronal differentiation inducer may be in the form of a salt, provided there is no loss in the neuronal differentiation-inducing activity.
  • use may be made of an acid addition salt of the peptide, which may be obtained by subjecting a commonly used inorganic acid or organic acid to an addition reaction according to a conventional method.
  • use may be made of other salts (e.g., metal salts), provided they have neuronal differentiation-inducing activities.
  • the neuronal differentiation inducer may also include, apart from the neuronal differentiation-inducing peptide serving as the active ingredient, various carriers that are medically (pharmaceutically) acceptable for the mode of use. Carriers that are generally used in peptide medications as diluents, excipients or the like are preferred. Although these may suitably differ according to the use and form of the neuronal differentiating inducer, typical examples include water, physiological buffers and various organic solvents.
  • the carrier may be an aqueous solution containing a suitable concentration of an alcohol (e.g., ethanol), glycerol, or a non-drying oil such as olive oil. Alternatively, the carrier may be liposomes.
  • secondary ingredients that may be included in the neuronal differentiation inducer include various fillers, thickeners, binders, wetting agents, surfactants, dyes and fragrances.
  • the form of the neuronal differentiating inducer is not subject to any particular limitation. Examples of typical forms include liquid preparations, suspensions, emulsions, aerosols, foams, pellets, powders, tablets, capsules and ointments.
  • the neuronal differentiating inducer may be rendered into a freeze-dried form or granules for preparing a drug solution by dissolution in physiological saline or a suitable buffer (e.g., PBS) just prior to use.
  • the process of preparing a drug (composition) in various forms by using as the materials the neuronal differentiation-inducing peptide (main ingredient) and various carriers (secondary ingredients) may itself be in general accordance with a conventional known method. Because such preparation processes themselves are not distinctive to the present invention, detailed descriptions are omitted here.
  • An example of a detailed information source relating to formulation is Comprehensive Medicinal Chemistry , edited by Corwin Hansch and published by Pergamon Press (1990). The entire contents of this book are incorporated herein by reference.
  • the neuronal differentiation inducer furnished by the present invention may be used in a manner and dose that accords with the form thereof and the intended purpose.
  • the neuronal differentiation-inducing peptide containing the APP signal peptide-related sequence disclosed herein may be administered as a liquid preparation to the patient (i.e., in vivo) in exactly the desired amount by intravenous, intramuscular, hypodermal, intradermal or intraperitoneal injection.
  • this neuronal differentiation-inducing peptide may be administered orally in a solid form such as tablets.
  • nerve cells can be generated (produced) from somatic stem cells present within the living organism, typically at or near the site of disease. This makes it possible to effectively treat various neurological disorders for which nerve regeneration is an important mode of treatment.
  • neurological disorders such as Alzheimer disease, Parkinson disease, cerebral infarction, paralysis of the body due to spinal cord injury, cerebral contusions, amyotrophic lateral sclerosis, Huntington disease, brain tumors and retinal degeneration by a regenerative medical approach is achieved.
  • nerve cells can be efficiently generated in vitro. This means that the desired nerve cells can be produced in a large quantity within such cellular material.
  • nerve cells that have been produced in a large quantity, or cellular material (living tissue or cell mass) containing these produced nerve cells are returned again to the living organism (typically, at the site of disease where neuronal regeneration is required), therapeutic effects similar to those obtained when a neuronal differentiation inducer (neuronal differentiation-inducing peptide) is administered directly in vivo are achievable.
  • a neuronal differentiation inducer neuronal differentiation-inducing peptide
  • this invention is also able to provide cells, cell masses or living tissue in which differentiation to nerve cells useful in the treatment of neurological disorders has been induced by using one of the neuronal differentiation-inducing peptides disclosed herein.
  • polynucleotides coding for the neuronal differentiation-inducing peptides of the invention may be used as materials employed in so-called gene therapy. For example, by integrating a gene (typically, a DNA segment or a RNA segment) coding for a neuronal differentiation-inducing peptide into a suitable vector and inserting the vector at the target site, it is possible to continuously express the neuronal differentiation-inducing peptide of the present invention within a living organism (cell). Therefore, polynucleotides (e.g., DNA segments, RNA segments) coding for the neuronal differentiation-inducing peptides of the present invention are useful as drugs for treating or preventing neurological disorders in the above types of patients.
  • a gene typically, a DNA segment or a RNA segment
  • coding for the neuronal differentiation-inducing peptides of the present invention are useful as drugs for treating or preventing neurological disorders in the above types of patients.
  • Table 1 The total of 15 types of peptides (Samples 1 to 15) shown in Table 1 were produced using the subsequently described peptide synthesizer. Table 1 shows the amino acid sequences and the total number of amino acid residues for each of the sample peptides.
  • each sample peptide is a chemically synthesized straight-chain peptide constituted so as to include on the C-terminal side of the peptide chain the nucleolar localization signal-related sequence shown in SEQ ID NO: 1 and to include on the N-terminal side thereof, after an intervening linker composed of one glycine residue, an amino acid sequence derived from the APP signal peptide shown in SEQ ID NO: 2 or SEQ ID NO: 3, and is composed of from 19 to 31 amino acid residues in all.
  • Sample 1 and Sample 2 each have, nearer the N-terminal with respect to the glycine linker, a partial amino acid sequence selected from the APP signal peptide sequence shown in SEQ ID NO: 2.
  • Sample 1 has, as the APP signal peptide-related sequence, an N-terminal side partial amino acid sequence composed of the total of 6 amino acid residues from the position 1 (N-terminal) methionine residue to the position 6 alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 2.
  • Sample 2 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 11 amino acid residues from the position 7 leucine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 2.
  • Sample 3 has, as the APP signal peptide-related sequence, the entire signal peptide sequence of SEQ ID NO: 3.
  • Samples 4 to 15 each have, on the N-terminal side from the glycine linker, a partial amino acid sequence selected from the APP signal peptide sequence shown in SEQ ID NO: 3.
  • Sample 4 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 15 amino acid residues from the position 3 proline residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 5 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 14 amino acid residues from the position 4 serine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 6 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 13 amino acid residues from the position 5 leucine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 7 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 12 amino acid residues from the position 6 alanine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 8 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 11 amino acid residues from the position 7 leucine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 9 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 10 amino acid residues from the position 8 leucine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 10 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 9 amino acid residues from the position 9 leucine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 11 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 8 amino acid residues from the position 10 leucine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 12 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 7 amino acid residues from the position 11 alanine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 13 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 6 amino acid residues from the position 12 alanine residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 14 has, as the APP signal peptide-related sequence, a C-terminal side partial amino acid sequence composed of the total of 5 amino acid residues from the position 13 tryptophan residue to the position 17 (C-terminal) alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3.
  • Sample 15 has an amino acid sequence composed of the total of 6 amino acid residues from the position 7 leucine residue to the position 12 alanine residue, counting from the N-terminal amino acid residue of the signal peptide sequence of SEQ ID NO: 3. That is, Sample 15 does not correspond to either the above-described N-terminal side partial amino acid sequence or the above-described C-terminal side partial amino acid sequence defined in the specification. Hence, Sample 15 is a peptide which does not have the APP signal peptide-related sequence defined in the specification.
  • Each of the above peptides was synthesized by a solid-phase synthesis process (Fmoc process) using a commercial peptide synthesizer (MultiPep RS, a product of Intavis AG).
  • HATU O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronioum hexafluorophosphate, a product of Watanabe Chemical Industries, Ltd.
  • DIEA diisopropylethylamine, a product of Wako Pure Chemical Industries, Ltd.
  • the resins and amino acids used in the solid-phase synthesis process were procured from NOVA Biochem.
  • Rink Amide resin 100 to 200 mesh
  • the deprotection reaction and condensation reaction were repeatedly carried out in accordance with the synthesis program of the peptide synthesizer, thereby elongating the peptide from the Fmoc-amino acid which bonds to the resin so as to obtain a synthesized peptide of the intended chain length.
  • the operations of cleaving and removing the Fmoc group serving as the amino protecting group on the amino acid with 20% piperidine/dimethylformamide (DMF) (peptide synthesis grade, a product of Wako Pure Chemical Industries), washing with DMF, reacting with 4 equivalents each of Fmoc-amino acid (—OH) and washing with DMF were repeated.
  • the Fmoc group was cleaved with 20% piperidine/DMF, and the reaction product was washed, first with DMF, then with ethanol.
  • the synthesized peptide chain was transferred together with the resin to a centrifuge tube, 1.8 mL of ethanediol, 0.6 mL of m-cresol, 3.6 mL of thioanisole and 24 mL of trifluoroacetic acid were added, and the mixture was stirred at room temperature for 2 hours. The resin that had been bonded to the peptide chain was then removed by filtration.
  • the resulting peptide precipitate was dried in vacuo, and purification was carried out using a high-performance liquid chromatograph (Waters 600, a product of Waters Corporation).
  • the molecular weights of each of the eluted peptides were determined based on matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF/MS) using the Voyager DE RPTM manufactured by PerSeptive Biosystems. As a result, the target peptide was confirmed to have been synthesized and purified.
  • Example 1 The neuronal differentiation-inducing activities of the synthesized peptides obtained in Example 1 (Samples 1 to 15) were examined.
  • the sample peptide was added to a culture broth of neuronal stem cells collected from a mouse (mouse neuronal stem cell growth medium, a product of Cell Applications), and incubated. Addition was carried out to a concentration of 0.5 ⁇ M for each peptide.
  • each of the cultured cells was nuclear stained with DAPI (4′,6-diamidino-2-phenylindole) and examined with a fluorescence microscope.
  • evaluation with a neuronal differentiation induction marker was carried out on the same samples. That is, using tubulin (specifically, ⁇ 3-tubulin) as the marker for identifying neurons (nerve cells), the presence of tubulin (i.e., the presence or absence of neurons) within the culture solution was checked by a fluorescence antibody method using a fluorochrome-labeled anti-tubulin antibody for tubulin identification. The results are shown in FIGS. 1 to 15 . The number of each figure corresponds to the number of the sample peptide used.
  • Each of these figures is a fluorescence micrograph (image) obtained by examining the state of neuronal differentiation by mouse neuronal stem cells following addition of the respective sample peptides and 7 days of culturing.
  • the image was prepared by merging a DIC image, a DAPI nuclear stain image, and a fluorescence image showing the results of an investigation by an immune antibody method using fluorochrome-labeled anti-tubulin antibody.
  • mouse neuronal stem cells were cultured in a mouse neuronal stem cell differentiation medium (product of Cell Applications) without adding any of the peptides.
  • a mouse neuronal stem cell differentiation medium product of Cell Applications
  • mouse neuronal stem cells were cultured in a mouse neuronal stem cell differentiation growth medium (product of Cell Applications) without adding any of the peptides.
  • a mouse neuronal stem cell differentiation growth medium product of Cell Applications
  • FIG. 16 As a result of the above evaluation tests, pronounced neuronal differentiation equal to or greater than the positive control ( FIG. 16 ) was observed when the artificial peptides of Samples 1 to 14 (neuronal differentiation-inducing peptides) were added (see FIGS. 1 to 14 ). That is, even in cases where the peptides of any of Samples 1 to 14 were added, fluorescence due to the presence of fluorochrome-labeled anti-tubulin antibody was clearly observed. Of these, the peptides of Samples 2, 6, 7 and 8 were observed to have particularly high neuronal differentiation-inducing activities. On the other hand, in a negative control using the same growth medium, such fluorescence (neuronal differentiation) was not observed ( FIG. 17 ).
  • neuronal stem cells differentiate into neurons owing to addition of the sample peptides, and thus demonstrates the usefulness of the APP signal peptide as a peptide motif relating to neuronal differentiation induction.
  • the Sample 1 peptide 50 mg
  • 50 mg of crystallized cellulose and 400 mg of lactose were mixed together, following which 1 mL of an ethanol/water mixture was added and kneading was carried out.
  • the kneaded material was then granulated according to a conventional method, thereby giving granules (granular neuronal differentiation inducer) containing a neuronal differentiation-inducing peptide as the main ingredient.
  • the neuronal differentiation-inducing peptides of the invention have high neuronal differentiation-inducing activities, and thus can be employed as peptide ingredients for medicinal purposes.
  • SEQ ID NO: 1 to SEQ ID NO: 35 Synthetic peptides

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